This proposal is a competing renewal of AI 33713. The long-term goal of this research is to determine the role of quorum sensing in the pathogenesis of Pseudomonas aeruginosa. We have shown that P. aeruginosa makes three signal molecules (autoinducers); PAI-1, PAI-2, and PQS. PAI-1 and PAI-2 activate the R-proteins LasR and RhlR respectively, to stimulate expression of numerous virulence factor and secondary metabolite genes. These two R proteins belong to the growing family of autoinducer responsive transcriptional regulators found in many Gram-negative bacteria. Homology searches of the genome show that P. aeruginosa contains two other R protein genes. Despite the advances made in understanding these important regulatory systems many questions remain. The current proposal seeks answers to some of these questions and has the following specific aims: 1. Continue studies on PAI-1 and PAI-2 by producing and characterizing polyclonal and monoclonal antibodies to these autoinducers. 2. Continue studies on the structure and function of LasR and RhlR. 3. Determine if the other two R proteins in P. aeruginosa PAO are involved in quorum sensing and the regulation of known virulence factors including proteases. 4. Continue studies on the role of LasR-PAI-1 and RhlR-PAI-2 in virulence of P. aeruginosa, using a model of acute pneumonia in adult mice and collaboratively in a mouse thermal injury model. We will examine the effect on bacterial virulence of mutations in QS genes and determine if antibodies to PAI-1 or PAI-2 alter the outcome of these P. aeruginosa infections. The results of these studies will further our understanding of virulence regulation in P. aeruginosa and provide new insights into quorum sensing systems in general. They will determine the feasibility of novel approaches to therapy for Gram-negative infections and provide useful reagents for future studies.